When nonpermissive mammalian cells are infected with avian sarcoma viruses (ASV) a small percentage of the infected cell population undergoes cellular transformation. A pivotal step in the establishment and maintenance of the transformed state is the formation of a DNA copy (provirus) of the viral RNA, and the subsequent integration of the proviral DNA into the host cell genome. This research proposal is designed to study the integration of avian proviral DNA in mammalian cells and, specifically, to determine the location and specificity of the integration site(s) on the viral and host cell genomes, and to examine the linear arrangement of the integratad viral genes in the transformed cell DNA. Using restriction endonucleases, preparative procedures for the separation and elution of DNA fragments, and high specific-activity cDNA probes, we will first establish a detailed restriction enzyme cleavage map of unintegrated form I ASV proviral DNA. We will then use a variety of ASV-transformed mammalian cell lines to establish the restriction enzyme cleavage pattern of integrated viral DNA sequences. These studies will allow us to determine if indeed there is a unique viral integration site in the different transformed cell lines. The hybridization of viral-specific DNA fragments to site-specific ASV cDNA probes will help us determine the linear arrangement of integrated viral genes in the various transformed cells. Utilizing restriction enzymology and specific hybridization we will examine the restriction enzyme cleavage pattern of acutely-infected nontransformed mammalian cells and revertants of transformed mammalian cells to determine the relationship between the cellular site(s) of viral DNA integration and the regulation of viral gene expression in these eukaryotic cell systems. These studies should provide some insight into the molecular mechanisms involved in the integration of a tumor virusgenome into mammalian cell DNa and, ultimately, into the molecular basis of the viral-mediated conversion of a normal cell into a transformed malignant cell.